Rational engineering of 2-deoxyribose-5-phosphate aldolases for the biosynthesis of (R)-1,3-butanediol

Kim, Taeho; Stogios, Peter J.; Khusnutdinova, Anna N.; Nemr, Kayla; Skarina, Tatiana; Flick, Robert; Joo, Jeong Chan; Mahadevan, Radhakrishnan; Savchenko, Alexei; Yakunin, Alexander F.

doi:10.1074/jbc.ra119.011363

Title: Rational engineering of 2-deoxyribose-5-phosphate aldolases for the biosynthesis of (R)-1,3-butanediol

Journal Article · 04 December 2019 · Journal of Biological Chemistry

DOI: https://doi.org/10.1074/jbc.ra119.011363 · OSTI ID:1657596

Kim, Taeho ^[1];

^[2]; Khusnutdinova, Anna N. ^[2]; Nemr, Kayla ^[2]; Skarina, Tatiana ^[2]; Flick, Robert ^[2]; Joo, Jeong Chan ^[3]; Mahadevan, Radhakrishnan ^[2]; Savchenko, Alexei ^[4];

^[2]

Univ. of Toronto, ON (Canada); LG Chem, Seoul (Korea)
Univ. of Toronto, ON (Canada)
Korea Research Inst. of Chemical Technology, Daejeon (Korea)
Univ. of Toronto, ON (Canada); Univ. of Calgary, AB (Canada)

Carbon–carbon bond formation is one of the most important reactions in biocatalysis and organic chemistry. In nature, aldolases catalyze the reversible stereoselective aldol addition between two carbonyl compounds, making them attractive catalysts for the synthesis of various chemicals. Here, we identified several 2-deoxyribose-5-phosphate aldolases (DERAs) having acetaldehyde condensation activity, which can be used for the biosynthesis of (R)-1,3-butanediol (1,3BDO) in combination with aldo-keto reductases (AKRs). Enzymatic screening of 20 purified DERAs revealed the presence of significant acetaldehyde condensation activity in 12 of the enzymes, with the highest activities in BH1352 from Bacillus halodurans, TM1559 from Thermotoga maritima, and DeoC from Escherichia coli. The crystal structures of BH1352 and TM1559 at 1.40–2.50 Å resolution are the first full-length DERA structures revealing the presence of the C-terminal Tyr (Tyr²²⁴ in BH1352). The results from structure-based site-directed mutagenesis of BH1352 indicated a key role for the catalytic Lys¹⁵⁵ and other active-site residues in the 2-deoxyribose-5-phosphate cleavage and acetaldehyde condensation reactions. These experiments also revealed a 2.5-fold increase in acetaldehyde transformation to 1,3BDO (in combination with AKR) in the BH1352 F160Y and F160Y/M173I variants. The replacement of the WT BH1352 by the F160Y or F160Y/M173I variants in E. coli cells expressing the DERA + AKR pathway increased the production of 1,3BDO from glucose five and six times, respectively. Thus, our work provides detailed insights into the molecular mechanisms of substrate selectivity and activity of DERAs and identifies two DERA variants with enhanced activity for in vitro and in vivo 1,3BDO biosynthesis.

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Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: Genome Canada; Natural Sciences and Engineering Research Council of Canada (NSERC); Ontario Research Fund

OSTI ID:: 1657596

Journal Information:: Journal of Biological Chemistry, Journal Name: Journal of Biological Chemistry Journal Issue: 2 Vol. 295; ISSN 0021-9258

Publisher:: American Society for Biochemistry and Molecular BiologyCopyright Statement

Country of Publication:: United States

Language:: ENGLISH

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Related Subjects

1
3-butanediol
2-deoxyribose-5-phosphate aldolase (DERA)
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
59 BASIC BIOLOGICAL SCIENCES
BH1352
Escherichia coli (E. coli)
acetaldehyde condensation
aldo-keto reductase
biotechnology
crystal structure
protein engineering
site-directed mutagenesis

Title: Rational engineering of 2-deoxyribose-5-phosphate aldolases for the biosynthesis of (R)-1,3-butanediol

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